The purpose of this project is to explore structure-function relationships in important phagocytic cell proteins. Current efforts are focused on the components of the NADPH oxidase, a system comprised of both membrane bound and soluble proteins. Interest in this system lies at two levels: 1) Studies on the the structure and function of this enzyme complex will address the molecular basis of a critical host defense mechanism of phagocytic white blood cells, since the NADPH oxidase is responsible for the generation of superoxide and its potent microbicidal oxygen metabolites. 2) The activation and assembly of the oxidase is thought to involve mechanisms common to other intercellular signal transduction systems relevant in all cells, since two of the oxidase components contain sequence motifs (SH3 domains) exhibiting significant homology to p60-src and other important intracellular proteins. We have recently cloned full length cDNAs encoding the four of oxidase factors p47-phox, p-67-phox, p22-phox, and gp91-phox) affected in Chronic Granulomatous Disease of Childhood (CGD) within a number of expression vectors. Two of these factors (p47-phox and p67-phox) have been purified to homogeneity from a recombinant baculovirus system, allowing detailed biochemical studies previously not feasible with the native proteins. These recombinant factors were used to restore cell-free NADPH oxidase activity of CGD patient cytosolic preparations to nearly normal levels. While the two recombinant proteins together were not sufficient to reconstitute the NADPH oxidase when mixed with membranes, a third soluble component (P28-phox) was identified and purified from crude cytosol based on its ability to complement the activities of recombinant p47-phox and p67-phox in a cell-free oxidase assay system. Finally, the roles of src-like sequence motifs in p67-phox have been explored by (deletion mutagenesis and reconstitution using the genetically modified recombinant proteins. These src-like sequences do not appear to be critical for oxidase activity, but are thought to function in cellular localization and activation processes.